Genetic and gene expression analysis of flowering time regulation by light quality in lentil.

BACKGROUND AND AIMS: Flowering time is important due to its roles in plant adaptation to different environments and subsequent formation of crop yield. Changes in light quality affect a range of developmental processes including flowering time, but little is known about light quality-induced flowering time control in lentil. This study aims to investigate the genetic basis for differences in flowering response to light quality in lentil. METHODS: We explored variation in flowering time caused by changes in red/far-red-related light quality environments of a lentil interspecific recombinant inbred line (RIL) population developed from a cross between Lens culinaris cv. Lupa and L. orientalis accession BGE 016880. A genetic linkage map was constructed and then used for identifying quantitative trait loci (QTLs) associated with flowering time regulation under different light quality environments. Differential gene expression analysis through transcriptomic study and RT-qPCR were used to identify potential candidate genes. KEY RESULTS: QTL mapping located 13 QTLs controlling flower time under different light quality environments, with phenotypic variance explained ranging from 1.7 to 62.9 %. Transcriptomic profiling and gene expression analysis for both parents of this interspecific RIL population identified flowering-related genes showing environment-specific differential expression (flowering DEGs). One of these, a member of the florigen gene family FTa1 (LcFTa1), was located close to three major QTLs. Furthermore, gene expression results suggested that two other florigen genes (LcFTb1 and LcFTb2), MADS-box transcription factors such as LcAGL6/13d, LcSVPb, LcSOC1b and LcFULb, as well as bHLH transcription factor LcPIF6 and Gibberellin 20 oxidase LcGA20oxC,G may also be involved in the light quality response. CONCLUSIONS: Our results show that a major component of flowering time sensitivity to light quality is tightly linked to LcFTa1 and associated with changes in its expression. This work provides a foundation for crop improvement of lentil with better adaptation to variable light environments.

Comprehensive hormone profiling of the developing seeds of four grain legumes.

KEY MESSAGE: Developmental context and species-specific hormone requirements are of key importance in the advancement of in vitro protocols and manipulation of seed development. Improvement of in vitro tissue and cell culture protocols in grain legumes such as embryo rescue, interspecific hybridization, and androgenesis requires an understanding of the types, activity, and balance of hormones within developing seeds. Towards this goal, the concentration of auxin, cytokinin, gibberellin, and abscisic acid (ABA) and their precursors and derivatives were measured in the developing seeds of field pea (Pisum sativum L.), chickpea (Cicer arietinum L.), lentil (Lens culinaris Medik.), and faba bean (Vicia faba L.) from 4 days after anthesis until 8 days after reaching maximum fresh weight. The importance of developmental context (developmental time and space) is demonstrated in both the differences and similarities between species for hormone profiles, especially with regard to cytokinin and ABA biosynthesis during the embryo formation. Auxin and its conjugates are significant during the pattern formation stage of all legumes; however, IAA-Asparagine appears important in the Vicieae species and its concentrations are greater than IAA from the globular stage of embryo development on in multi-seed fruits. Finally, the significance of non-polar gibberellins during lentil seed development is highlighted.

Multifaceted roles of transcription factors during plant embryogenesis.

Transcription factors (TFs) are diverse groups of regulatory proteins. Through their specific binding domains, TFs bind to their target genes and regulate their expression, therefore TFs play important roles in various growth and developmental processes. Plant embryogenesis is a highly regulated and intricate process during which embryos arise from various sources and undergo development; it can be further divided into zygotic embryogenesis (ZE) and somatic embryogenesis (SE). TFs play a crucial role in the process of plant embryogenesis with a number of them acting as master regulators in both ZE and SE. In this review, we focus on the master TFs involved in embryogenesis such as BABY BOOM (BBM) from the APETALA2/Ethylene-Responsive Factor (AP2/ERF) family, WUSCHEL and WUSCHEL-related homeobox (WOX) from the homeobox family, LEAFY COTYLEDON 2 (LEC2) from the B3 family, AGAMOUS-Like 15 (AGL15) from the MADS family and LEAFY COTYLEDON 1 (LEC1) from the Nuclear Factor Y (NF-Y) family. We aim to present the recent progress pertaining to the diverse roles these master TFs play in both ZE and SE in Arabidopsis, as well as other plant species including crops. We also discuss future perspectives in this context.

Androgenesis-inducing stress treatments change phytohormone levels in anthers of three legume species (Fabaceae).

UNLABELLED: Legumes are recalcitrant to androgenesis and induction protocols were only recently developed for pea (Pisum sativum L.) and chickpea (Cicer arietinum L.), albeit with low regeneration frequencies. Androgenesis is thought to be mediated through abscisic acid (ABA) but other phytohormones, such as auxins, cytokinins, and gibberellins, have also been implicated. In view of improving induction protocols, the hormone content of pea, chickpea, and lentil anthers was measured after exposure to cold, centrifugation, electroporation, sonication, osmotic shock, or various combinations thereof using an analytical mass spectrometer. Indole-3-acetic acid (IAA) had a key function during the induction process. In pea, high concentrations of IAA-asparagine (IAA-Asp), a putative IAA metabolite, accumulated during the application of the different stresses. In chickpea, the IAA-Asp concentration increased 30-fold compared to pea but only during the osmotic shock treatment and likely as a result of the presence of exogenous IAA in the medium. In contrast, no treatment in lentil (Lens culinaris) invoked such an increase in IAA-Asp content. Of the various cytokinins monitored, only cis zeatin riboside increased after centrifugation and electroporation in pea and possibly chickpea. No bioactive gibberellins were detected in any species investigated, indicating that this hormone group is likely not linked to androgenesis in legumes. In contrast to the other stresses, osmotic shock treatment caused a reduction in the levels of all hormones analyzed, with the exception of IAA-Asp in chickpea. A short period of low hormone content might be a necessary transition phase for androgenesis induction of legumes. KEY MESSAGE: Five androgenesis-inducing stress treatments changed content of ABA, auxin and cytokinin in anthers of three legumes. Osmotic shock treatment differed because it reduced hormone content to very low levels.

RNA-Seq and Gene Ontology Analysis Reveal Differences Associated With Low R/FR-Induced Shade Responses in Cultivated Lentil and a Wild Relative.

Lentil is an important pulse crop not only because of its high nutrient value but also because of its ecological advantage in a sustainable agricultural system. Our previous work showed that the cultivated lentil and wild lentil germplasm respond differently to light environments, especially to low R/FR-induced shade conditions. Little is known about how cultivated and wild lentils respond to shade at the level of gene expression and function. In this study, transcriptomic profiling of a cultivated lentil (Lupa, L. culinaris) and a wild lentil (BGE 016880, L. orientalis) at several growth stages is presented. De novo transcriptomes were assembled for both genotypes, and differential gene expression analysis and gene ontology enrichment analysis were performed. The transcriptomic resources generated in this study provide fundamental information regarding biological processes and genes associated with shade responses in lentils. BGE 016880 and Lupa shared a high similarity in their transcriptomes; however, differential gene expression profiles were not consistent between these two genotypes. The wild lentil BGE 016880 had more differentially expressed genes than the cultivated lentil Lupa. Upregulation of genes involved in gibberellin, brassinosteroid, and auxin synthesis and signaling pathways, as well as cell wall modification, in both genotypes explains their similarity in stem elongation response under the shade. Genes involved in jasmonic acid and flavonoid biosynthesis pathways were downregulated in BGE 016880 only, and biological processes involved in defense responses were significantly enriched in the wild lentil BGE 016880 only. Downregulation of WRKY and MYB transcription factors could contribute to the reduced defense response in BGE 016880 but not in Lupa under shade conditions. A better understanding of shade responses of pulse crop species and their wild relatives will play an important role in developing genetic strategies for crop improvement in response to changes in light environments.

[Correlation Between Microplastics Pollution and Eutrophication in the Near Shore Waters of Dianchi Lake].

Microplastics have been found in many environmental media such as sea water, coastal tidal flats, terrestrial water, sediments, and organisms. Microplastics pollution in inland freshwater lakes have received extensive attention; however, the correlation between eutrophication and microplastics pollution in freshwater lakes remains unclear. In this study, 24 sampling sites were set up in the near shore surface waters of Dianchi Lake, and the pollution characteristics of microplastics such as abundance, composition, particle size, color, and form were evaluated. Water quality parameters related to eutrophication state were analyzed, and the eutrophication indices were further calculated. Specifically, sample pre-treatment was conducted according to the method issued by National Oceanic and Atmospheric Administration (NOAA) of the United States. The color and morphological characteristics of microplastic samples were observed using a stereoscopic microscope, and counts and particle size measurements were performed using Nano Measure 1.2 software. Parts of the samples were selected, and the polymer composition analysis was performed using micro-Fourier Transform infrared (µ-FTIR) spectroscopy. The indices related to eutrophication level evaluation were tested according to the experimental standard methods issued by the Ministry of Ecology and Environment of China. The results showed that the abundance of microplastics in the near shore waters of Dianchi Lake was between 800 and 6000 n·m-3, with an average value of 2867 n·m-3. The types of polymers detected were polyethylene terephthalate (PET), polyetherurethane (PEU), polypropylene (PP), polyethylene (PE), and polyvinyl acetate (PVAc), respectively. The diameter proportion of microplastics in the range of 0.2-0.5 mm was the highest. Fiber microplastics accounted for the most observed type, followed by fragments and films. Among the 24 monitoring sites, it was found that proportions of severe, moderate, and mild eutrophication and mesotrophication sites accounted for 8.33%, 58.33%, 29.17%, and 4.17% of the total sampling sites, respectively, and the main pollutant was total nitrogen (TN). Microplastics abundances in the near shore waters of Dianchi Lake were significantly positively correlated with TN concentrations (P<0.01), whereas they were negatively correlated with chlorophyll a(Chl-a)concentrations, not reaching a significant level (P>0.05). The microplastics abundance and TN concentrations in the north bank water near the main urban area of Kunming were significantly higher than those in the other three banks. Microplastics and TN were considered to potentially have the same origin and be attributed to the tail water discharge from WWTPs.

An active part of Artemisia sacrorum Ledeb. attenuates hepatic lipid accumulation through activating AMP-activated protein kinase in human HepG2 cells.

Artemisia sacrorum Ledeb. (Compositae) (ASL) is a traditional Chinese medicine used to treat different hepatic diseases. However, a hypolipidemic effect of ASL on fatty liver disease has not been reported. Therefore, we investigated whether 95% ethanol eluate (EE), an active part of ASL, would attenuate hepatic lipid accumulation in human HepG2 cells by activating AMP-activated protein kinase (AMPK). Significant decreases in triglyceride levels and increases in AMPK and acetyl-CoA carboxylase (ACC) phosphorylation were observed when the cells were treated with 95% EE. EE down-regulated the lipogenesis gene expression of sterol regulatory element-binding protein 1c (SREBP1c) and its target genes, such as fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1), in a time- and dose-dependent manner. In contrast, the lipolytic gene expression of peroxisome proliferator-activated receptor alpha (PPAR-alpha) and CD36 increased in a time- and dose-dependent manner. These effects were abolished by pretreatment with compound C, an AMPK inhibitor. However, there were no differences in the gene expression of SREBP2, low density lipoprotein receptor (LDLR), hydroxymethyl glutaryl CoA reductase (HMG-CoA), or glucose transporter 2 (GLUT2). At the same time, 95% EE significantly increased the gene expression of acyl CoA oxidase (ACOX) in a time- and dose-dependent manner. Thus, AMPK mediated 95% EE induced suppression of SREBP1c and activation of PPAR-alpha respectively. These finding indicate that 95% EE attenuates hepatic lipid accumulation through AMPK activation and may be active in the prevention of serious diseases such as fatty liver, obesity, and type-2 diabetic mellitus.

[Expressions of LEAFY homologous genes in different organs and stages of Ginkgo biloba].

Expressions of Ginlfy and GinNdly gene were studied by northern blotting in different organs and stages of Ginkgo Biloba. Ginlfy gene was expressed in different organs such as root, stem, leaf of juvenile tree, male tree and female tree, and in different stages of male flower bud and female flower bud. It was inferred that Ginlfy gene could be expressed constitutionally. GinNdly gene was only expressed in leaf of juvenile tree, male tree and female tree and in different stages of male flower bud and female flower bud, while GinNdly gene was not expressed in the other organs. Therefore it was thought that GinNdly gene could be expressed differentially and be a close relation to development of flower.

Classification and characterization of species within the genus lens using genotyping-by-sequencing (GBS).

Lentil (Lens culinaris ssp. culinaris) is a nutritious and affordable pulse with an ancient crop domestication history. The genus Lens consists of seven taxa, however, there are many discrepancies in the taxon and gene pool classification of lentil and its wild relatives. Due to the narrow genetic basis of cultivated lentil, there is a need towards better understanding of the relationships amongst wild germplasm to assist introgression of favourable genes into lentil breeding programs. Genotyping-by-sequencing (GBS) is an easy and affordable method that allows multiplexing of up to 384 samples or more per library to generate genome-wide single nucleotide Polymorphism (SNP) markers. In this study, we aimed to characterize our lentil germplasm collection using a two-enzyme GBS approach. We constructed two 96-plex GBS libraries with a total of 60 accessions where some accessions had several samples and each sample was sequenced in two technical replicates. We developed an automated GBS pipeline and detected a total of 266,356 genome-wide SNPs. After filtering low quality and redundant SNPs based on haplotype information, we constructed a maximum-likelihood tree using 5,389 SNPs. The phylogenetic tree grouped the germplasm collection into their respective taxa with strong support. Based on phylogenetic tree and STRUCTURE analysis, we identified four gene pools, namely L. culinaris/L. orientalis/L. tomentosus, L. lamottei/L. odemensis, L. ervoides and L. nigricans which form primary, secondary, tertiary and quaternary gene pools, respectively. We discovered sequencing bias problems likely due to DNA quality and observed severe run-to-run variation in the wild lentils. We examined the authenticity of the germplasm collection and identified 17% misclassified samples. Our study demonstrated that GBS is a promising and affordable tool for screening by plant breeders interested in crop wild relatives.

Molecular variations in Th1-specific cell surface gene Tim-3.

The family of T-cell immunoglobulin domain and mucin domain (TIM) proteins is identified to be expressed on T cells. A member of Tim family, Tim-3 (T cell immunoglobulin mucin 3) is selectively expressed on the surface of differentiated Th1 cells. Tim-3 might have an important role in the induction of autoimmune diseases by regulating macrophage activation and interacts with Tim-3 ligand to regulate Th1 responses. To determine the variation sites in the coding and promoter region of human Tim-3 gene, we performed variation scanning by direct sequencing using the genomic DNA isolated from the patients with asthma or allergic rhinitis and healthy controls without asthma and allergic rhinitis. We identified four single nucleotide polymorphisms (SNPs) including one novel SNPs (-1541C>T) and two variation sites (-1292_-1289delTAAA and -1282_-1278dupTAAAA) in the coding and promoter region of human Tim-3 gene in both the patients and healthy groups.